The invention relates to the field of organ transplantation.
Technical advances in allogeneic organ transplantation and the availability of nonspecific immunosuppressive agents have revolutionized the field of organ transplantation. This progress has, however, resulted in a shortage of essential organs of suitable size and match.
The shortage of allograft-organs has led to an increased interest in xenogeneic transplantation. It was demonstrated more than twenty-five years ago that transplants from chimpanzee to man could provide long-term life-supporting function. However, the use of non-human primates as an organ source is of limited applicability. Many primate species are scarce and protected, and those that are more plentiful, such as the baboon, often do not grow to a size which allows the use of their organs in adults. Moreover, in some cultures, the use of primates as a source of organs is ethically unacceptable.
Some of these difficulties could be resolved by use of ungulate organs, especially pig organs. Pigs are domesticated, easy to breed, have large litters, and grow rapidly to the size which allow the use of their organs in the very largest human beings. In addition, pig and man have many anatomical and physiological similarities. However, transplantation of a pig organ into a human results in a vigorous rejection of the graft-organ.
In general, the invention features, a genetically engineered swine cell, e.g., a cultured swine cell, a retrovirally transformed swine cell, or a cell derived from a transgenic swine. The cell includes a transgene which encodes a xenogeneic, e.g., a human, class I MHC protein, e.g., an HLA A, B, C or G gene.
In preferred embodiments the transgene includes an xcex1 subunit, e.g., an HLA class I gene, e.g., an HLA C gene.
Where the transgene includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. As is discussed below, alleles of HLA class I genes can often be classed into reactivity groups wherein an allele from a reactivity group can confer protection against NK cells specific to other alleles in the reactivity group. Thus, in preferred embodiments, the transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has: an Asn at residue 77 and a Lys at residue 80; or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments the transgene includes an HLA A gene. In other preferred embodiments the transgene includes an HLA B gene.
In other preferred embodiments the transgene includes an HLA G gene, e.g, any of alleles I-IV of HLA G.
In preferred embodiments: the cell includes a second transgene which includes a class I MHC protein. In preferred embodiments the second transgene includes an HLA class I gene, e.g., an HLA A, B, C or G gene. In preferred embodiments the first transgene includes an allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. For example, the first transgene includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In preferred embodiments the first transgene encodes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene encodes an allele which has a Ser at residue 77 and an Asn at residue 80. In other preferred embodiments the second transgene encodes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene.
In preferred embodiments the transgene includes a chimeric class I gene, e.g., a chimeric HLA A, B, C, or G gene. The chimeric transgene can include a first portion derived from a first allele of a gene encoding a class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80, see e.g., Biassoni, 1995, J. Exp. Med. Vol. 182: 605-609, hereby incorporated by reference. See also Moretta et al., 1996, Ann. Rev. Immunol. 14: 619-648, hereby incorporated by reference, which together with the disclosure herein, provides guidance for altering critical residues in the HLA C genes.
In yet other preferred embodiments the genetically engineered swine cell is: a swine hematopoietic stem cell, e.g., a cord blood hematopoietic stem cell, a bone marrow hematopoietic stem cell, or a fetal or neonatal liver or spleen hematopoietic stem cell; derived from differentiated blood cells, e.g. a myeloid cell, such as a megakaryocyte, monocyte, granulocyte, or an eosinophil; an erythroid cell, such as a red blood cell, e.g. a lymphoid cell, such as B lymphocytes and T lymphocytes; derived from a pluripotent hematopoietic stem cell, e.g. a hematopoietic precursor, e.g. a burst-forming units-erythroid (BFU-E), a colony forming unit-erythroid (CFU-E), a colony forming unit-megakaryocyte (CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM), a colony forming unit-eosinophil (CFU-Eo), or a colony forming unit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swine cell other than a hematopoietic stem cell, or other blood cell; a swine thymic cell, e.g., a swine thymic stromal cell; a bone marrow stromal cell; a swine liver cell; a swine kidney cell; a swine epithelial cell; a swine hematopoietic progenitor cell; a swine muscle cell, e.g., a heart cell; an endothelial cell; or a dendritic cell or precursor thereof.
In yet other preferred embodiments the cell is: isolated or derived from cultured cells, e.g., a primary culture, e.g., a primary cell culture of hematopoietic stem cells; isolated or derived from a transgenic animal.
In yet other preferred embodiments: the swine cell is homozygous for the transgene; the swine cell is heterozygous for the transgene; the swine cell is homozygous for the transgene (heterozygous transgenic swine can be bred to produce offspring that are homozygous for the transgene); the swine cell includes two or more transgenes.
In yet other preferred embodiments the cell includes a one or more, or all of, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, and a transgene which encodes an HLA G gene.
In another aspect, the invention features a nucleic acid, e.g., a transgene, including a swine promoter operably linked to a xenogeneic, e.g., human, nucleic acid which encodes a class I MHC protein. The swine promoter can be, e.g., a swine hematopoietic epithelial gene promoter, or a heterologous inducible or developmentally regulated promoter.
In preferred embodiments the nucleic acid includes, e.g., a gene which encodes an a subunit, e.g., an HLA class I gene, e.g., an HLA A, B, C, or G gene, or a gene which encodes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene
Where the nucleic acid includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the nucleic acid encodes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments, the nucleic acid or transgene further includes transcriptional regulatory sequences, e.g. a tissue-specific promoter, e.g., a hematopoietic specific promoter, operably linked to the non-swine gene sequence.
In preferred embodiments the nucleic acid or transgene encodes a chimeric class I protein, e.g., a chimeric HLA A, B, C, or G protein. The chimeric transgene can include a first portion derived from a first allele of a gene encoding a class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene is a chimeric or mutated HLA C gene which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In another aspect, the invention features, a transgenic swine having cells which include a xenogeneic, e.g., a human, nucleic acid, e.g., a transgene which encodes an HLA class I protein. In preferred embodiments the transgene can include a nucleic acid which encodes an a subunit, e.g., an HLA class I gene, e.g., one or more of. In other preferred embodiments the transgene includes a nucleic acid which encodes a human xcex2 subunit, e.g. xcex2-2 microglobulin gene.
In preferred embodiments in which the transgene includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments the transgene encodes a chimeric class I protein, e.g., chimeric HLA A, B, C, or G gene. The chimeric transgene can include a first portion derived from a first allele of a gene encoding a class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In preferred embodiments: the transgenic swine includes a second transgene which encodes a class I MHC protein. In preferred embodiments the transgene includes a gene which encodes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene. In other preferred embodiments the second transgene includes an HLA class I gene, e.g., an HLA A, B, C, or G gene. In preferred embodiments the first transgene includes an allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. In preferred embodiments the transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the first transgene encodes an HLA C allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene encodes an HLA C allele which has a Ser at residue 77 and an Asn at residue 80.
In yet other preferred embodiments: the transgenic swine cell is hemizygous for the transgene; the transgenic swine cell is hemizygous for the transgene; the transgenic swine is heterozygous for the transgene; the transgenic swine is homozygous for the transgene (heterozygous transgenic swine can be bred to produce offspring that are homozygous for the transgene); the transgenic swine includes two or more transgenes.
In yet other preferred embodiments the transgenic swine includes a one or more, or all, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, a transgene which encodes an HLA G gene.
Transgenic swine (or swine cells) of the invention can be used as a source for xe2x80x9chumanizedxe2x80x9d tissue for grafting into a human recipient, e.g., hematopoietic cells or other tissues or organs.
In another aspect, the invention features, a swine organ or a swine tissue, having cells which include a xenogeneic, e.g., a human, nucleic acid, e.g., a transgene which encodes an HLA class I protein. In preferred embodiments the transgene can include a nucleic acid which encodes an a subunit, e.g., an HLA class I gene, e.g., an HLA A, B, C, or G gene. In other preferred embodiments the transgene includes a nucleic acid which encodes a human xcex2 subunit, e.g. xcex2-2 microglobulin gene.
In preferred embodiments the organ is a heart, lung, kidney, pancreas, or liver.
In preferred embodiments the tissue is: thymic tissue; islet cells or islets; stem cells; bone marrow; endothelial cells; skin; or vascular tissue.
In preferred embodiments in which the transgene includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments the transgene encodes a chimeric class I protein, e.g., chimeric HLA A, B, C, or G gene. The chimeric transgene can include a first portion derived from a first allele of a gene encoding a class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In yet other preferred embodiments: the transgenic swine organ or tissue includes: a cell which is hemizygous for the transgene; a cell which is hemizygous for the transgene; a cell which is heterozygous for the transgene; a cell which is homozygous for the transgene (heterozygous transgenic swine can be bred to produce offspring that are homozygous for the transgene); a cell which includes two or more transgenes.
In preferred embodiments: the swine organ or tissue includes cells which include a second transgene which encodes a class I MHC protein. In preferred embodiments the second transgene includes a gene which encodes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene. In other preferred embodiments the second transgene includes an HLA class I gene, e.g., an HLA A, B, C, or G gene. In preferred embodiments the first transgene includes an allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. In preferred embodiments the first transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the first transgene includes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene includes an allele which has a Ser at residue 77 and an Asn at residue 80.
In yet other preferred embodiments the organ or tissue includes one or more, or all, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, a transgene which encodes an HLA G gene.
The swine organs and tissues of the invention can be used as a source for xe2x80x9chumanizedxe2x80x9d tissue for grafting into a human recipient, e.g., hematopoietic cells or other tissues or organs.
Graft tissue which expresses a recipient species-MHC class I gene can be used to improve methods of transplanting xenogeneic tissue into a recipient. For example, acceptance of porcine tissue by a human recipient can be prolonged if the porcine tissue expresses a human class I gene, preferably the HLA C gene. The use of graft tissue which expresses an HLA class I gene can be combined with methods of inducing tolerance described herein.
Accordingly, in another aspect, the invention features a method of inducing tolerance in a recipient mammal, e.g., a primate, e.g., a human, to a graft from a swine, e.g., a miniature swine. The method includes:
inserting DNA encoding a swine MHC antigen, preferably a class I antigen, a class II antigen, or both, into a hematopoietic stem cell, e.g., a bone marrow hematopoietic stem cell, of the recipient mammal;
allowing the MHC antigen encoding DNA to be expressed in the recipient; and
implanting the graft in the recipient, wherein some or substantially all of the cells of the graft express a recipient species class I gene which inhibits recipient NK cell mediated attack.
In preferred embodiments the graft tissue expresses an HLA C gene. The allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the allele is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments the graft expresses an HLA A gene. In other preferred embodiments the graft expresses an HLA B gene. In other preferred embodiments the graft expresses an HLA G gene, e.g, any of alleles I-IV of HLA G. In yet other preferred embodiments the graft includes one or more, or all, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, a transgene which encodes an HLA G gene.
In preferred embodiments: the graft includes a first transgene which includes an HLA class I allele, e.g., an HLA C allele, and a second transgene which includes a gene which encodes a class I MHC protein. In preferred embodiments the second transgene includes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene. In other preferred embodiments the first transgene includes an HLA class I allele from a first reactivity group and the second transgene includes an allele from the second reactivity group. For example, the first transgene includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In preferred embodiments the first transgene encodes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene encodes an allele which has a Ser at residue 77 and an Asn at residue 80. In other preferred embodiments the second transgene encodes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene.
In preferred embodiments the graft expresses a chimeric class I protein, e.g., chimeric HLA A, B, C, or G gene. For example, the chimeric gene includes a first portion derived from a first allele of the gene encoding the class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In preferred embodiments the graft is from a transgenic swine, e.g., a transgenic swine which includes a transgenic human class I gene, e.g., an HLA A, B, C, or G gene.
In preferred embodiment, the method further includes: administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine treatment. The short course of help reducing treatment is generally administered at about the time the graft is introduced into the recipient. The short course of help reducing treatment can induce tolerance to unmatched class I and/or minor antigens on a graft which is introduced into the recipient subsequent to expression of the MHC antigen. The duration of the short course of help reducing treatment should be approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments, the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen. In other preferred embodiments, the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of a steroid drug in a sufficient concentration to counteract the desired effect of the help reducing treatment, e.g., in the absence of Prednisone (17, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione) at a concentration which stimulates the release of a cytokine by mature T cells in the recipient. In preferred embodiments, the short course of help reducing treatment is administered in the absence of a steroid drug, e.g., in the absence of Prednisone. In preferred embodiments: the help reducing treatment is begun before or at about the time the graft is introduced; the short course is perioperative; or the short course is postoperative.
Preferred embodiments include those in which: the recipient stem cell is removed from the recipient mammal prior to the DNA insertion and returned to the recipient mammal after the DNA insertion; the DNA is obtained from the individual mammal from which the graft is obtained; the DNA is obtained from an individual mammal which is syngeneic with the individual mammal from which the graft is obtained; the DNA is obtained from an individual mammal which is MHC matched, and preferably identical, with the individual mammal from which the graft is obtained; the DNA includes an MHC class I gene; the DNA includes an MHC class II gene; the DNA is inserted into the cell by transduction, e.g., by a retrovirus, e.g., by a Moloney-based retrovirus; and the DNA is expressed in bone marrow cells and/or peripheral blood cells of the recipient for at least 14, preferably 30, more preferably 60, and most preferably 120 days, after the DNA is introduced into the recipient.
Other preferred embodiments include: the step of, prior to hematopoietic stem cell transplantation, creating hematopoietic space, e.g., by irradiating the recipient mammal with low dose, e.g., between about 100 and 400 rads, whole body irradiation to deplete or partially deplete the bone marrow of the recipient; inactivating thymic T cells by one or more of: prior to hematopoietic stem cell transplantation, irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation, or administering to the recipient a short course of an immunosuppressant, as is described herein.
Other preferred embodiments include: the step of, prior to implantation of a graft, depleting natural antibodies from the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the second species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.)
In other preferred embodiments: the method further includes, prior to hematopoietic stem cell transplantation, introducing into the recipient an antibody capable of binding to mature T cells of said recipient mammal.
Other preferred embodiments further include the step of introducing into the recipient a graft obtained from the donor, e.g., a liver or a kidney.
In preferred embodiments: the donor graft cells are other than a hematopoietic stem cells, or other blood cells; the donor graft cells are swine thymic cells, e.g., swine thymic stromal cells; the donor graft cells are bone marrow stromal cells; the donor graft cells are swine liver cells; the donor graft cells are swine kidney cells; the donor graft cells are swine epithelial cells; the donor graft cells are swine muscle cells, e.g., heart cells; the donor graft cells are swine neuronal cells; the graft cells include an organ, e.g., a kidney, a liver, or a heart; the donor graft cells include dendritic cells or their precursors.
Other preferred embodiments include: the step of introducing into the recipient, donor species-specific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus. In preferred embodiments: the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells.
In another aspect, the invention features a method of inducing tolerance in a recipient mammal, e.g., a primate, e.g., a human, to a graft obtained from a donor swine, e.g., a miniature swine. The method includes:
preferably prior to or simultaneous with transplantation of the graft, introducing, e.g., by intravenous injection, into the recipient mammal, swine hematopoietic stem cells, e.g., bone marrow cells or fetal liver or spleen cells (preferably the hematopoietic stem cells home to a site in the recipient mammal);
(optionally) inactivating the natural killer cells of the recipient mammal, e.g., by prior to introducing the hematopoietic stem cells into the recipient mammal, introducing into the recipient mammal an antibody capable of binding to natural killer cells of said recipient mammal; and
implanting the graft in the recipient, provided that one or both of: the stem cells, or some or substantially all of the cells of the graft, express a recipient species class I gene which inhibits recipient NK cell mediated attack.
In preferred embodiments either or both, a stem cell or the graft tissue, expresses an HLA C allele. The allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the allele is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments either or both, a stem cell or the graft tissue, expresses an HLA A gene. In other preferred embodiments either or both, a stem cell or the graft tissue, graft expresses an HLA B gene. In other preferred embodiments either or both, a stem cell or the graft tissue, expresses an HLA G gene, e.g, any of alleles I-IV of HLA G. In preferred embodiments either or both, a stem cell or the graft tissue, includes one or more, or all, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, a transgene which encodes an HLA G gene.
In preferred embodiments: either or both, a stem cell or the graft tissue, includes a first transgene which includes an HLA class I allele, e.g., an HLA C allele, and a second transgene which includes a gene which encodes a class I MHC protein. In preferred embodiments the second transgene includes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene. In other preferred embodiments the first transgene includes an HLA class I allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. For example, the first transgene includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the first transgene includes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene includes an allele which has a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments either or both, a stem cell or the graft tissue, expresses a chimeric class I protein, e.g., chimeric HLA A, B, C, or G gene. For example, the chimeric gene includes a first portion derived from a first allele of the gene encoding the class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In preferred embodiments either or both, a stem cell or the graft tissue, is from a transgenic swine, e.g., a transgenic swine which includes a transgenic human class I gene, e.g., an HLA A, B,C, or G gene.
In preferred embodiment, the method further includes: administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine treatment. The short course of help reducing treatment is generally administered at about the time the graft is introduced into the recipient. The short course of help reducing treatment can induce tolerance to unmatched class I and/or minor antigens on a graft which is introduced into the recipient subsequent to expression of the MHC antigen. The duration of the short course of help reducing treatment should be approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments, the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen. In other preferred embodiments, the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of a steroid drug in a sufficient concentration to counteract the desired effect of the help reducing treatment, e.g., in the absence of Prednisone (17, 21-dihydroxypregna-1,4-diene-3, 11, 20-trione) at a concentration which stimulates the release of a cytokine by mature T cells in the recipient. In preferred embodiments, the short course of help reducing treatment is administered in the absence of a steroid drug, e.g., in the absence of Prednisone. In preferred embodiments: the help reducing treatment is begun before or at about the time the graft is introduced; the short course is perioperative; or the short course is postoperative.
The hematopoietic cells prepare the recipient for the graft that follows, by inducing tolerance at both the B-cell and T-cell levels. Preferably, hematopoietic cells are fetal liver or spleen, or bone marrow cells, including immature cells (i.e., undifferentiated hematopoietic stem cells; these desired cells can be separated out of the bone marrow prior to administration), or a complex bone marrow sample including such cells can be used.
One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum. As is discussed below, preferably, a second anti-mature T cell antibody can be administered as well, which lyses T cells as well as NK cells. Lysing T cells is advantageous for both bone marrow and xenograft survival. Anti-T cell antibodies are present, along with anti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses of anti-NK or anti-T cell antibody may be preferable. Monoclonal preparations can be used in the methods of the invention.
Other preferred embodiments include: the step of introducing into the recipient mammal, donor species-specific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus. In preferred embodiments: the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells; the hematopoietic stem cells are introduced simultaneously with, or prior to, the antibody.
Other preferred embodiments include those in which: the same mammal of the second species is the donor of one or both the graft and the hematopoietic cells; and the antibody is an anti-human thymocyte polyclonal anti-serum, obtained, e.g., from a horse or pig.
Other preferred embodiments include: the step of, prior to hematopoietic stem cell transplantation, creating hematopoietic space, e.g., by irradiating the recipient mammal with low dose, e.g., between about 100 and 400 rads, whole body irradiation to deplete or partially deplete the bone marrow of the recipient; inactivating thymic T cells by one or more of: prior to hematopoietic stem cell transplantation, irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation, or administering to the recipient a short course of an immunosuppressant, as is described herein.
Other preferred embodiments include: the step of, prior to hematopoietic stem cell transplantation, depleting natural antibodies from the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the second species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.)
In other preferred embodiments: the method further includes, prior to hematopoietic stem cell transplantation, introducing into the recipient an antibody capable of binding to mature T cells of said recipient mammal.
In other preferred embodiments: the method further includes inactivating T cells of the recipient, e.g., by, prior to introducing the hematopoietic stem cells into the recipient, introducing into the recipient an antibody capable of binding to T cells of the recipient.
In preferred embodiments, the method includes the step of introducing into the recipient a graft obtained from the donor which is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
In preferred embodiments: the donor graft cells are other than a hematopoietic stem cells, or other blood cells; the donor graft cells are swine thymic cells, e.g., swine thymic stromal cells; the donor graft cells are bone marrow stromal cells; the donor graft cells are swine liver cells; the donor graft cells are swine kidney cells; the donor graft cells are swine endothelial cells; the donor graft cells are swine epithelial cells; the donor graft cells are swine muscle cells, e.g., heart cells; the donor graft cells are swine neuronal cells; the graft cells include an organ, e.g., a kidney, a liver, or a heart; the donor graft cells include dendritic cells or their precursors.
Other preferred embodiments include: the step of introducing into the recipient, donor species-specific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus. In preferred embodiments: the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells.
Genetically engineered swine cells of the invention can be made by methods known to those skilled in the art, e.g., by retroviral transduction of swine cells. Methods for producing transgenic swine of the invention use standard transgenic technology. These methods include, e.g., the infection of the zygote or organism by viruses including retroviruses; the infection of a tissue with viruses and then reintroducing the tissue into an animal; and the introduction of a recombinant nucleic acid molecule into an embryonic stem cell of a mammal followed by appropriate manipulation of the embryonic stem cell to produce a transgenic animal. In particular, the invention features a transgenic swine, whose germ cells and somatic cells contain a transgene including a DNA sequence encoding a polypeptide and a tissue-specific promoter operably linked to the DNA sequence, wherein the tissue-specific promoter effects expression of the hematopoietic peptide in bone marrow cells of the swine, the transgene being introduced into embryonal cells of the animal, or an ancestor of the animal.
In another aspect, the invention features, a method of inducing tolerance in a recipient mammal, e.g., a primate, e.g., a human, to a graft obtained from a swine, e.g., a miniature swine. The method includes:
prior to or simultaneous with transplantation of the graft, introducing into the recipient mammal, swine thymic tissue, e.g., thymic epithelium, preferably fetal or neonatal thymic tissue; and
implanting the graft in the recipient. The thymic tissue prepares the recipient for the graft that follows, by inducing immunological tolerance at the T-cell level. Either or both of, the thymic tissue, or some or all of the cells of the graft, express a recipient species class I gene which inhibits recipient NK cell mediated attack.
In preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, expresses an HLA C allele. The allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the allele is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, expresses an HLA A gene. In other preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, expresses an HLA B gene. In other preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, expresses an HLA G gene, e.g, any of alleles 14V of HLA G. In preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, includes one or more, or all, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, a transgene which encodes an HLA G gene
In preferred embodiments: either or both of, the thymic tissue, or some or all of the cells of the graft, includes a first transgene which includes an HLA class I allele, e.g., an HLA C allele, and a second transgene which includes a gene which encodes a class I MHC protein. In preferred embodiments the second transgene includes a human xcex2 subunit, e.g., a xcex2-2 microglobulin gene. In other preferred embodiments the first transgene includes an HLA class I allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. For example, the first transgene includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the first transgene includes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene includes an allele which has an a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, expresses a chimeric class I protein, e.g., chimeric HLA A, B, C, or G gene. For example, the chimeric gene includes a first portion derived from a first allele of the gene encoding the class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80.
In preferred embodiments either or both of, the thymic tissue, or some or all of the cells of the graft, is from a transgenic swine, e.g., a transgenic swine which includes a transgenic human class I gene, e.g., an HLA A, B, C, or G gene.
Preferred embodiments include other steps to promote acceptance of the graft thymus and the induction of immunological tolerance or to otherwise optimize the procedure. In preferred embodiments: liver or spleen tissue, preferably fetal or neonatal liver or spleen tissue, is implanted with the thymic tissue; donor hemopoietic cells, e.g., cord blood stem cells or fetal or neonatal liver or spleen cells, are administered to the recipient, e.g., a suspension of fetal liver cells administered intraperitoneally or intravenously; the recipient is thymectomized, preferably before or at the time the xenograft thymic tissue is introduced.
In other preferred embodiments the method includes (preferably prior to or at the time of introducing the thymic tissue or stem cells into the recipient) depleting, inactivating or inhibiting recipient NK cells, e.g., by introducing into the recipient an antibody capable of binding to natural killer (NK) cells of the recipient, to prevent NK mediated rejection of the thymic tissue; (preferably prior to or at the time of introducing the thymic tissue into the recipient) depleting, inactivating or inhibiting recipient T cells, e.g., by introducing into the recipient an antibody capable of binding to T cells of the recipient; (preferably prior to or at the time of introducing the thymic tissue or stem cells into the recipient) depleting, inactivating or inhibiting host CD4+ cell function, e.g., by introducing into the recipient an antibody capable of binding to CD4, or CD4+ cells of the recipient. An anti-mature T cell antibody which lyses T cells as well as NK cells can be administered. Lysing T cells is advantageous for both thymic tissue and xenograft survival. Anti-T cell antibodies are present, along with anti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses of anti-NK or anti-T cell antibody may be preferable. Monoclonal preparations can be used in the methods of the invention.
Other preferred embodiments include those in which: the recipient does not receive hemopoietic cells from the donor or the donor species: the same mammal of the second species is the donor of both the graft and the thymic tissue; the donor mammal is a swine, e.g., a miniature swine; an anti-human thymocyte polyclonal anti-serum, obtained, e.g., from a horse or pig is administered to the recipient.
Other preferred embodiments include the step of (preferably prior to thymic tissue or hematopoietic stem cell transplantation) creating hematopoietic space, e.g., by one or more of: irradiating the recipient mammal with low dose, e.g., between about 100 and 400 rads, whole body irradiation, the administration of a myelosuppressive drug, the administration of a hematopoietic stem cell inactivating or depleting antibody, to deplete or partially deplete the bone marrow of the recipient.
Other preferred embodiments include (preferably prior to thymic tissue or hematopoietic stem cell transplantation) inactivating thymic T cells by one or more of: irradiating the recipient with, e.g., about 700 rads of thymic irradiation, administering to the recipient one or more doses of an anti T cell antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonal antibody, or administering to the recipient a short course of an immunosuppressant.
In preferred embodiments the host or recipient is a post-natal individual, e.g., an adult, or a child.
In preferred embodiments the method further includes the step of identifying a host or recipient which is in need of a graft.
Other preferred embodiments include depleting or otherwise inactivating natural antibodies, e.g., by one or more of: the administration of a drug which depletes or inactivates natural antibodies, e.g., deoxyspergualin; the administration of an anti-IgM antibodies; or the absorption of natural antibodies from the recipient""s blood, e.g., by contacting the hosts blood with donor antigen, e.g., by hemoperfusion of a donor organ, e.g., a kidney or a liver, from the donor species.
As is discussed herein, killer inhibitory receptors (KIR""s) found on NK cells are specific for polymorphic MHC class I molecules. Swine tissue used for grafts should express an HLA class I allele (or alleles) which will maximize protection from the NK cells of the recipient. Thus, in another aspect, the invention includes a method of prolonging acceptance of a swine xenograft tissue by a human which includes:
determining the HLA A, HLA B, HLA C, or HLA G phenotype or genotype of the recipient;
choosing donor tissue, e.g., choosing a donor transgenic animal, which expresses a recipient HLA gene which will confer protection against recipient NK cells and implanting the tissue in the recipient. For example, the donor tissue or animal can express a transgenic form of an allele expressed by the recipient. Alternatively, the donor tissue or animal can express an allele from the same reactivity group as one or more of the alleles expressed in the recipient.
The recipient""s HLA A, B, C, or G phenotype or genotype can be determined by standard methods, e.g., by PCR or lymphocyte toxicity.
In another aspect, the invention includes, a panel of transgenic swine including a first transgenic swine and a second transgenic swine. The first transgenic swine has a transgene which includes a first allele of a human HLA A, B, C, or G gene, and the second transgenic swine has a transgene which includes a second allele of the human HLA A, B, C, or G gene. In preferred embodiments the first allele is from a first reactivity group and the second allele is from a second reactivity group. For example, the transgene of the first swine includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the transgene of the second swine includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments transgene of the first swine includes an allele which has an Asn at residue 77 and a Lys at residue 80 and the transgene of the second swine includes an allele which has an a Ser at residue 77 and an Asn at residue 80. The panel of transgenic swine can be used to supply a donor having an HLA class I allele which will protect a graft from a recipient""s NK cells.
NK cells play an important role in the rejection of xenogeneic tissues. Because NK cell killing is inhibited by the presence of class I antigens of the NK cell type on the target cells, NK-mediated attack of porcine tissues by human NK cells is minimized by the invention. This is achieved by introducing a human class I gene into the porcine target.
In contrast to allogeneic cell-mediated killing, xenogeneic human anti-porcine cytotoxicity includes an important contribution from NK cells. Normal autologous and most allogeneic cells are not susceptible to natural killer (NK) cell mediated cytotoxicity due to the expression of major histocompatibility complex (MHC) class I molecules. The preventive signal is delivered to the NK cells through KIR""s with different MHC class I specificities. Xenogeneic porcine MHC class I molecules appear not to be recognized by human KIR""s and thus render porcine cells susceptible to NK cell-mediated lysis.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.